The broad, long-term objective of this R21 CEBRA application is to advance the overall understanding of molecular mechanisms regulating endogenous cannabinoid signaling by identifying proteins responsible for endocannabinoid uptake. The endogenous cannabinoids such as anandamide are fatty acid-derived molecules whose metabolism and signal termination appear to rely upon transport back into releasing cells for subsequent breakdown by an intracellular amidohydrolase enzyme. Thus, the uptake process is thought to play a critical role in the dynamic regulation of endogenous cannabimimetic activity. The Specific Aim of this proposal is to identify proteins involved with the uptake and intracellular transport of the endocannabinoid anandamide. This project has direct health-relatedness as the endogenous cannabinoid system has been implicated in modulating various physiological and pathological processes including mood, anxiety, psychosis, motor movement, inflammation, blood pressure, and pain. Recent evidence suggests that "lipid raft-" or caveolae-related endocytosis may be involved with internalization and cellular transport of anandamide. Thus, a carrier protein for anandamide would be predicted to reside in this membrane microdomain. By developing novel probes and using cutting-edge proteomic methods, putative anandamide binding proteins will be identified. The role of these proteins in anandamide uptake will be explored using modern molecular, biochemical, and pharmacological approaches. These studies will provide a basis for future studies examining the molecular mechanisms associated with both structure/function and regulation of endogenous cannabinoid transport.